The ability of T cells to distinguish self from non-self remains one of the central paradigms of the immune system. This specialized capability begins early in T cell development and under normal circumstances is maintained throughout the life of an individual. Self- tolerance is maintained by peripheral as well as central (thymic) mechanisms. The primary goal of this proposal is to study the role of the CD28/CTLA-4/B7 family of co-stimulatory molecules in the regulation of peripheral tolerance. Studies performed during the previous program project grant identified the B7-1 and B7-2 molecules as major co-stimulatory ligands; demonstrated that CD28 signaling regulates sustained T cell proliferative responses and Th1/Th2 differentiation in naive T cells. Of equal importance, we demonstrated that CTLA-4/B7 interactions profoundly down-regulate T cell responses. This application proposes to take advantage of in vitro and in vivo model systems to define the roles of B7-1 and B7-2 in the regulation of both CD28 and CTLA-4-mediated functions; to study the biochemical basis of CTLA-4-mediated down-regulation of immunity; and to develop novel regulatory drugs to manipulate the immunological activities of these receptors. In order to accomplish these goals, the following specific aims are proposed. 1. Determine the mechanism of tolerance induction and the individual roles of B7-1 and B7-2 on donor and host tissues during the initiation and progression of transplant rejection; 2. Determine the biological effects of CTLA-4 engagement and blockade on exposure to antigen in vitro and in vivo; and 3. Determine the molecular and biochemical basis of CTLA-4-mediated suppression of T cell function in vitro. The results of these studies will provide important insights into the mechanisms of CD28/CTLA-4 regulation of immune responses and may lead to novel immunotherapeutic approaches to the induction and maintenance of peripheral tolerance.